Reappraisal of serum retinol-binding protein as a surrogate marker for retinol and discovery of a novel retinol estimation formula.

BACKGROUND & AIMS: Serum retinol (ROH) is commonly used for population level assessment of vitamin A status. High-performance liquid chromatography (HPLC) is considered most accurate method for measuring ROH. However, with the technical difficulty of using HPLC for routine assays, serum retinol-binding protein (RBP) measured by immunological assays is expected to be a surrogate marker for ROH, with reports of a close correlation between serum RBP and ROH. Nevertheless, RBP is not commonly tested to assess vitamin A status with concerns over RBP alterations under various physiopathological conditions. Thus, we reappraised the extent to which RBP could be used as a surrogate marker in representative disorders that alter serum RBP levels. As a related marker, diagnostic utility of transthyretin (TTR) was also evaluated. METHODS: To evaluate the reliability of ROH and RBP assays, specimen stability was assessed in terms of (1) storage at 25, 4, -20, and -80 °C for 1-28 days, (2) five-cycle freeze-thawing, and (3) fluorescent light exposure for 1-14 days. Sources of variation (sex, age, body mass index [BMI], and drinking habits) and reference intervals for ROH, RBP, and TTR were determined in 617 well-defined healthy individuals. To investigate the influence of disorders that affect serum RBP, patients with five diagnostic groups were enrolled: 26 with chronic kidney disease (CKD); 13 with various malignancies in advanced stages (AdM), 12 with acute bacterial infections (ABI), 6 with liver cirrhosis (LC), and 26 with simple obesity (BMI ≥ 27 kg/m2). RESULTS: The stability of RBP and ROH in serum was confirmed under all conditions. In healthy individuals, serum ROH, RBP, and TTR were appreciably high in males with a slight increase in proportion to age and BMI. The major-axis regression line between RBP (x) and ROH (y) in healthy individuals was y = x, with a correlation coefficient of 0.986. In the LC, AdM, and ABI groups, similar strong correlations were observed; however, the regression lines were shifted slightly rightward from the healthy group line, indicating a positive bias in estimating ROH. Interestingly, the same analyses between TTR and ROH revealed similar strong linear relationships in all groups; however, the regression line of each group showed a leftward (opposite) shift from the healthy group line. Based on these observations, we developed a novel regression model composed of RBP and TTR, which gave much improved accuracy in estimating ROH, even under these pathological conditions. CONCLUSIONS: The perfect RBP-ROH correlation in healthy individuals indicates the utility of RPB as a surrogate marker for ROH. Nevertheless, under RBP-altered conditions, a slight overestimation of ROH is inevitable. However, when the TTR was tested together, the bias can be corrected almost perfectly using the novel ROH estimation formula comprising RBP and TTR.

Does Retinol-Binding Protein 4 (holo- and apo-RBP4) Increase or Remain Constant in Sera from Patients with Hypothyroidism?

Retinol-binding protein 4 (RBP4) is a retinol transporter in the blood plasma. Many diseases alter the plasma or serum levels of RBP4. Since serum RBP4 concentrations have been reported to decrease in hyperthyroidism, this study investigated whether serum RBP4 concentrations increased or remained constant in hypothyroidism. In sera from patients with hypothyroidism (n=71), hyperthyroidism (n=30), and healthy subjects (n=20), serum concentrations of RBP4 (sum of holo- and apo-RBP4), retinol, albumin, creatinine, and related constituents were measured, and RBP4/retinol molar ratio (as an index of apo-RBP4) and estimated glomerular filtration rate (eGFR) were calculated. The results showed that serum RBP4 concentrations tended to increase with decreasing free thyroxine concentrations, but there were no significant differences among patients with hypothyroidism, hyperthyroidism, and healthy subjects. When patients with hypothyroidism were subdivided by serum RBP4 level using 2.1 µmol/L cut-off value, patients with >2.1 µmol/L were revealed to be patients with older age having lower tri-iodothyronine, higher holo-RBP4, higher apo-RBP4, higher retinol, higher RBP4/retinol molar ratio, and lower eGFR than those in patients with <2.1 µmol/L. Multiple regression analysis showed significant associations between serum RBP4 levels and explanatory variables (retinol and eGFR). Although serum levels of RBP4 prior to the onset of renal dysfunction may affect the present concentrations, we conclude that the increase of serum RBP4 (both holo- and apo-RBP4) in patients with hypothyroidism was attributed to the decline in eGFR. In contrast, serum RBP4 concentration remained constant without renal dysfunction.

A Case of ß-Carotenemia Misdiagnosed as Jaundice by the Bilirubin Oxidase Method.

Serum bilirubin measurement is necessary to accurately distinguish jaundice from carotenemia. A 59.8-y old Japanese male showed symptoms of yellow skin pigmentation as a result of ß-carotenemia. Diagnostic laboratory results indicated elevated levels of serum muscle enzymes (aspartate aminotransferase, lactate dehydrogenase, and creatine kinase), but normal levels in liver function tests (alanine aminotransferase and direct bilirubin). The laboratory results indicated hypothyroid myopathy. Moreover, although the patient did not show significant abnormalities in liver function tests, the serum level of total bilirubin (TBIL) measured by bilirubin oxidase method was markedly increased beyond the upper limit of normal. Fundamental experiments revealed that the bilirubin oxidase method had a positive interference by ß-carotene. These findings suggested that hyper ß-carotenemia could have caused the falsely elevated serum TBIL levels in the patient.

Surveillance evaluation of the standardization of assay values for serum total 25-hydroxyvitamin D concentration in Japan.

Background To assess the vitamin D nutritional status, serum total 25-hydroxyvitamin D (25(OH)D) concentration is measured. We used six automated 25(OH)D immunoassays (AIAs) available in Japan and certified by the Vitamin D Standardization Program (VDSP) at the U.S. Center for Disease Control and Prevention to assess the concordance of the assay results. Methods Serum total 25(OH)D concentrations in SRM 972a and 20 serum samples from patients were determined using three liquid chromatography-tandem mass spectrometry (LC-MS/MS) and six AIAs (pilot study), and an additional 110 serum samples were assessed by the six AIAs (surveillance study). The assay bias from the results of LC-MS/MS by Chiba University or consensus values (i.e. average of six AIAs) was estimated using the procedure described in CLSI document EP09-A3. Results LC-MS/MS at Chiba University could completely separate 25(OH)D2, 25(OH)D3 and 3-epi-25(OH)D3, and the observed values including total 25(OH)D in SRM 972a were all within ±1·SD of the assigned values. All AIAs produced results greater than ±3·SD. In the pilot study, four of the six AIAs had an average percentage bias, as estimated by confidence interval (CI), larger than ±5% (acceptance criterion in CLSI); the bias converged from -6.5% to 3.2% after adjustment by LC-MS/MS. In the surveillance study, 25(OH)D concentrations in AIAs all adjusted to LC-MS/MS converged within ±5% from consensus values. However, some AIAs showed negative or positive bias from the consensus values. Conclusions Current AIAs in Japan continue to lack standardization. Manufacturers should implement quality assurance strategies so that their values more closely align to those of standard reference material 972a.

Anti-diabetic effects of lactic acid bacteria in normal and type 2 diabetic mice.

The antidiabetic effects of lactic acid bacteria were investigated using mice. In Experiment 1, normal ICR mice were loaded with sucrose or starch with or without viable Lactobacillus rhamnosus GG cells. GG significantly inhibited postprandial blood glucose levels when administered with sucrose or starch. In Experiment 2, KK-A(y) mice, a model of genetic type 2 diabetes, were given a basal diet containing viable GG cells or viable Lactobacillus delbrueckii subsp. bulgaricus cells for 6 weeks. Viable GG cells significantly inhibited fasting blood glucose, postprandial blood glucose in a glucose tolerance test and HbA1c. Such effects were not shown by viable L. bulgaricus cells. In Experiment 3, the KK-A(y) mice were given a basal diet containing viable GG cells or heat-treated GG cells for 3 weeks. The viable GG cells significantly suppressed fasting blood glucose and impaired glucose tolerance, but the heat-treated GG showed no effects. These results demonstrated that GG decreased the postprandial blood glucose in ICR mice, and that the antidiabetic activity of lactic acid bacteria on the KK-A(y) mice differed depending on the bacterial strain and whether the bacterium is viable when it arrives in the intestine. In the present study, we conclude that the antidiabetic activity may result from continuous inhibition of the postprandial blood glucose through suppression of glucose absorption from the intestine. These findings indicate that specific strains of lactic acid bacterium can be expected to be beneficial for the management of type 2 diabetes.

Commutability of National Institute of Standards and Technology standard reference material 1955 homocysteine and folate in frozen human serum for total folate with automated assays.

BACKGROUND: The aim of the present study was to evaluate standard reference material (SRM) 1955 commutability as a reference material for serum folate using automated methods. We also designed so as to reduce the intermethod variability present in different automated methods. METHODS: Using a microbiological assay related to the 'information value' of SRM 1955 as a comparison method, we investigated the possibility of standardization for the assay values of serum folate as measured by the automated methods (Access, Centaur and Elecsys). In the assay of 50 patient sera by these automated methods, we corrected observed values by the SRM 1955 and compared with comparison values. RESULTS: The observed values of SRM 1955 Levels I, II and III were within or outside (but near) a 95% prediction interval obtained from patient sera by the automated methods. The normalized residuals obtained from SRM 1955 were within ±3.0 (in SD units), which enabled us to conclude that the SRM 1955 had a physicochemical characterization similar to native serum. Twelve patients were assessed as hypofolataemia (<6.0 ng/mL) and 38 patients as normal (≥6.0 ng/mL). Before correction, folate levels in six of 12 patients were lower than 6.0 ng/mL, and those in seven of 38 patients were higher than 6.0 ng/mL with the automated methods. After correction, low levels were found in four of 12 patients, and normal levels were found in 33 of 38 patients. CONCLUSIONS: The use of SRM 1955 would help to reduce the intermethod variability present in different automated methods for serum folate measurement.

Estimation of vitamin B1 excretion in 24-hr urine by assay of first-morning urine.

Urinary B1 (vitamin B1) excretion is commonly determined in 24-hr urine specimens to obtain an estimate of nutritional status. The aim of our study was to investigate whether B1 in random urine specimens, corrected for the urine creatinine (Cr), can be substituted for B1 in 24-hr urines. Collection of such hour urines is often fraught with errors; an alternative method is described here. All urine specimens voided over 24 hr were collected from 32 healthy adults as were the first-morning urines from 30 healthy Japanese women. The B1 excretion was expressed as the ratio of B1 to Cr. Although the B1 excretion was expressed as the B1/Cr ratio, the B1 excretion varied with the urine volume and the time of urine collection. The B1/Cr ratio in random urine specimens not collected at a fixed time may mislead the evaluation of the nutritional status. We found that the B1/Cr ratio in the first-morning urine correlated significantly with the ratio in 24-hr urines (r=0.970, P<0.001) and also with the concentration of total B1 (B1 plus its phosphate esters) in whole blood (r=0.733, P<0.001). We conclude that the B1/Cr ratio in 24-hr urines could be estimated by measuring the ratio in the first-morning urine.

A system for nutritional consulting using quick questionnaires on diet and unidentified complaints.

The aging of society and ongoing health care cost-control policy set the trend for the self-medication which leads to the growing interest in health promotion and prolongation of healthy life expectancy through self-health management. We developed a self-medication support system to provide comprehensive support to consumers at pharmacies and drug stores. This system facilitates the effective use of information and knowledge based on medicine and health. This self-medication support system comprised a set of two terminals connected network server in the data center: a user terminal for consumers use and an advisor terminals for specialized advisor, pharmacists, registered dieticians, and etc. This system enables specialized sales people to provide the appropriate advice based on the factors of consumer's problem, and to make suggestions for improving his/her lifestyle: eating habit, doing exercise, and having relaxation time. As a result of the trial use of this system at pharmacy stores, a certain degree of correlation between the results of a questionnaire on unidentified complaints and dietary patterns causing potential micronutrient deficiency was demonstrated.

Effects of dietary ascorbic acid supplementation on lipid peroxidation and the lipid content in the liver and serum of magnesium-deficient rats.

We investigated the effects of ascorbic acid (AsA) supplementation on lipid peroxidation and the lipid content in the liver and serum of magnesium (Mg)-deficient rats. Eighteen 3-week-old male Sprague-Dawley strain rats were divided into 3 groups and maintained on a control diet (C group), a low-Mg diet (D group), or a low-Mg diet supplemented with AsA (DA group) for 42 d. At the end of this period, the final body weight, weight gain, and serum Mg concentrations were significantly decreased in the Mg-deficient rats. Further, dietary AsA supplementation had no effect on the growth, serum Mg concentration, Mg absorption, and Mg retention. The serum concentration of AsA was significantly lower in the D group than in the C group but was unaltered in the DA group. The levels of phosphatidylcholine hydroperoxide (PCOOH) in the serum and of triglycerides (TGs) and total cholesterol (TC) in the serum and liver were significantly higher in the D group than in the C group. The serum PCOOH, liver TG, and liver TC levels were decreased in the DA group. These results indicate that Mg deficiency increases the AsA requirement of the body and that AsA supplementation normalizes the serum levels of PCOOH and the liver lipid content in Mg-deficient rats, without altering the Mg status.

Beneficial effects of grape seed extract on malondialdehyde-modified LDL.

Following consecutive 12-wk administration of tablets containing 0, 200 or 400 mg grape seed extract (calculated as proanthocyanidin) to 61 healthy subjects with LDL cholesterol (LDL-C) levels of 100 to 180 mg/dL, effects of such treatment compared to administration of placebo tablets on malondialdehyde-modified LDL (MDA-LDL), representing one oxidized type of LDL, were investigated by a single blind method. MDA-LDL level in the 200 mg (calculated as proanthocyanidin) group was significantly (p = 0.008) reduced compared to the basal level, 12 wk after the start of administration. In the 400 mg (calculated as proanthocyanidin) group, a significant decrease in MDA-LDL level compared to the basal level was found 6 and 12 wk after the start of administration (6 wk: p = 0.015, 12 wk: p = 0.009). Subjects with high levels of MDA-LDL/ApoB (MDA-LDL/ApoB > or = 100 mU/mL) in the 200 mg group showed significantly (p = 0.011) reduced MDA-LDL levels at 12 wk after the start of administration. In the 400 mg group, significant decreases in MDA-LDL level compared to the basal level were seen 6 and 12 wk after the start of administration (6 wk: p = 0.001, 12 wk: p < 0.001); and at week 6, significantly (p = 0.048) lower values were observed compared to those in patients who took placebo tablets (0 mg proanthocyanidin). In subjects demonstrating the least body weight changes during the test period (less than +/- 1.0 kg) in the 400 mg group, there was an increasing trend (p = 0.088) in adiponectin levels 12 wk after the start of treatment. These results suggested that tablets containing grape seed extract exerted reducing effects on oxidized LDL, and might be useful in preventing lifestyle-related diseases such as arteriosclerosis.

Diurnal variation of biopyrrin excretion in random urine specimens is not corrected by creatinine.

Circulating bilirubin is thought to function as a physiological antioxidant. One of the decomposition products of this process is the biopyrrins, which include two regioisomers: biotripyrrin-a (1,14,15,17-tetrahydro-2,7,13-trimethyl-1,14-deoxy-3-vinyl-16H-tripyrrin-8,12-dipropionic acid) and biotripyrrin-b (1,14,15,17-tetrahydro-3,7,13-trimethyl-1,14-deoxy-3-vinyl-16H-tripyrrin-8,12-dipropionic acid). We measured biopyrrins in random urine specimens and investigated whether the biopyrrin values obtained were valid when expressed as a ratio of the creatinine (Cr) concentrations. All of the random urine specimens collected over 48 hr were from presumably healthy adults. We measured the biopyrrins by means of an enzyme-linked immunosorbent assay (ELISA) using an anti-bilirubin monoclonal antibody. When the values were expressed in terms of the ratio to Cr, the within-day coefficient of variation (%CV) of the excretion of biopyrrins was reduced to 27%+/-10% (P<0.05) from 59%+/-27%. However, assay values on random or spot urine specimens were unreliable because of the large %CV. The biopyrrin concentrations only in the first-morning-urine specimens in terms of both absolute amounts and ratios to Cr significantly reflected those in a 24-hr urine specimen (P<0.001). Concentrations in a random urine specimen voided at the second collection or later did not correlate with the concentration in a 24-hr urine specimen (P>0.05), even if their values were corrected by Cr. The amounts of biopyrrins excreted in 24-hr urine specimens were significantly correlated with the 24-hr cortisol excretion (P<0.001) but not to uropepsin (P>0.05).

Assay values for thiamine or thiamine phosphate esters in whole blood do not depend on the anticoagulant used.

We compared the whole blood, plasma, and erythrocyte (red blood cell (RBC)) concentrations of thiamine and thiamine phosphate esters in the presence of heparin or EDTA as anticoagulants. Three blood specimens were collected from each of 24 healthy volunteers into evacuated collection tubes containing the following anticoagulants: heparin, Na2EDTA, or K2EDTA. The concentrations of nonphosphorylated free thiamine (T), thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP) were determined by the NH2-column HPLC method. The anticoagulant used had no effect on the concentrations obtained in whole blood and plasma of thiamine or any of the above thiamine compounds (P>0.05). RBCs were isolated by centrifugation and washed with isotonic saline, and the cell counts of the washed cells were adjusted to their whole blood values. In the washed RBCs with any anticoagulant, the concentrations of T, TMP, and TDP expressed either as nmol/L of whole blood or a ratio to hemoglobin were significantly lower (P<0.05) than those in whole blood.

Extended-spectrum beta-lactamase-producing Shiga toxin gene (Stx1)-positive Escherichia coli O26:H11: a new concern.

Escherichia coli strain TUM2139 was isolated from a stool sample from a 9-year-old girl on 16 June 2004. This strain was categorized as Shiga toxin-producing Escherichia coli (STEC) because the Shiga-like toxin gene stx(1) was detected by immunochromatography and PCR assay. The strain was highly resistant to cefotaxime (256 microg/ml) and was also resistant to cefepime, cefpodoxime, ceftriaxone, and aztreonam. In the presence of 4 microg of clavulanic acid per ml, the MIC of cefotaxime decreased to < or =0.12 microg/ml, indicating that this strain was an extended-spectrum beta-lactamase (ESBL) producer. Cefotaxime resistance was transferred to E. coli C600 by conjugation at a frequency of 3.0 x 10(-6). A PCR assay was performed with primer sets specific for TEM-type and SHV-type ESBLs and for the CTX-M-2 (Toho-1), CTX-M-3, and CTX-M-9 groups of ESBLs. A specific signal was observed with the primer set specific for the CTX-M-9 group of beta-lactamases. This beta-lactamase was confirmed to be the ESBL CTX-M-18 by DNA sequencing. This is the first report of an ESBL-producing STEC isolate.

Values of water-soluble vitamins in blood and urine of Japanese young men and women consuming a semi-purified diet based on the Japanese Dietary Reference Intakes.

We investigated the levels of water-soluble vitamins except for vitamin B6 in the blood and urine of Japanese college male (n = 10) and female (n = 10) students. They consumed for 7 d a semi-purified diet based on Japanese Dietary Reference Intakes to assess the Recommended Dietary Allowances (RDA) for water-soluble vitamins and to present some new normal values for blood and urine levels of water-soluble vitamins in Japanese. The blood and the 24-h urine samples were collected on the last day of the experiment and measured. The values of total vitamin B1 in whole blood, total vitamin B2 in whole blood, total cyanocobalamin in serum, total nicotinamide in whole blood, total pantothenic acid in whole blood, total folates in serum, total biotin in serum, and ascorbic acid in plasma were 104+/-17 pmol/mL (mean+/-SD), 216+/-25 pmol/mL, 0.34+/-0.05 pmol/mL, 59.1+/- 5.0 nmol/ mL, 2.45+/-0.37 nmol/mL, 15.6+/-4.6 pmol/mL, 8.3+/-0.5 pmol/mL, and 62+/-10 nmol/mL, respectively, in males, and 90+/-23, 234+/-18, 0.67+/-0.20, 61.9+/-6.0, 2.48+/-0.30, 30.2+/-8.6, 8.4+/-0.3, and 67+/-14, respectively, in females. There was a significant difference in the values of cyanocobalamin and total folates between men and women. The urinary excretion of vitamin B1, vitamin B2, cyanocobalamin, sum of the catabolic metabolites of nicotinamide, pantothenic acid, folates, biotin, and ascorbic acid were 665+/-114 nmol/d, 562+/-325 nmol/d, 93+/-31 pmol/d, 84+/-26 micromol/d, 9.3+/-2.3 micromol/d, 19.4+/-2.8 nmol/d, 83+/-18 pmol/d, and 148+/-51 micromol/d, respectively, in males, and 495+/-212, 580+/-146, 145+/-49, 83+/-19, 16.9+/-1.3, 22.7+/-2.7, 83+/-23, and 140+/-51, respectively, in females. There was a significant difference in the urinary excretion of cyanocobalamin, pantothenic acid and total folates between men and women. These values will be useful for the nutritional assessment of water-soluble vitamins for Japanese, although the examination period was short. In future, an experiment with various age groups and re-evaluation by repeated experiments will provide more accurate values.

Recommended dietary allowance for vitamin C in the United States is also applicable to a population of young Japanese women.

The recommended dietary allowance (RDA) for ascorbic acid (AA) in Canada and the United States has been set for several years at 75 mg/day for women 19-30 years old. Recently this level was questioned, and an increase to 90 mg/day was suggested. For Japanese women in the same age group, we found that the RDA for AA is currently 100 mg/day. Our goal was to determine which RDA is sufficient for maintaining a serum concentration of AA in young Japanese women above the lower reference limit of 7.0 mg/L. We measured serum AA concentrations by an ascorbate oxidase method in 176 healthy Japanese women (19-26 years old). We also performed an ROC analysis to estimate the optimal cutoff value for oral dosage to distinguish individuals with hypovitaminosis-C (<7.0 mg/L) from those with a normal serum AA. We evaluated the Japanese RDA using the 75 or 90 mg/day U.S. RDA and the weight ratio between Japanese and U.S. women, and discovered that the RDA value ranged between 66 and 79 mg/day. From the ROC analysis, we found that the optimal daily dosage of AA is approximately 75 mg/day. This value gave the highest efficiency, sensitivity, negative predictive value, and positive likelihood ratio, and the lowest negative likelihood ratio. Therefore, an RDA of 100 mg/day may be unnecessarily high for young Japanese women.

Stability of fat-soluble and water-soluble vitamins in artificially prepared, vitamin-enriched, lyophilized serum.

Vitamin-enriched, lyophilized serum (VES) was prepared for an inter-laboratory study to compare vitamin assays. The VES contained water-soluble vitamins (vitamin B1, vitamin B12, vitamin C, and folate), fat-soluble vitamins (vitamin A and vitamin E), and cholesterol. We performed stability studies and determined vitamin concentrations and total cholesterol in VES stored at -20 degrees C for 12 months. Our recovery of the water-soluble vitamins in reconstituted VES was 70-142%, but we recovered only 33-45% of the fat-soluble vitamins. Physicochemical properties, such as specific gravity and viscosity of the reconstituted VES did not affect manual or automated measurements of these vitamins. Vial-to-vial differences found for the VES were the same as the within-day analytical variations. There was no evidence of degradation of vitamin A, vitamin B1, vitamin B12, vitamin C, folate, and cholesterol over 12 months in VES stored at -20 degrees C. Following deproteinization, vitamin C concentration was found to be lower than when not deproteinated. Vitamin E was less stable in VES, however, and the degradation during 12 months was lower than the between-day analytical variation of the assay. Our VES is the first preparation of lyophilized control serum that contains water-soluble and fat-soluble vitamins.

Antioxidant capacities of ascorbic acid, uric acid, alpha-tocopherol, and bilirubin can be measured in the presence of another antioxidant, serum albumin.

Human serum contains several antioxidants. The total antioxidant capacity (AOC) is the sum of all the antioxidant activities present in serum. The aim of our study was to investigate whether the AOC of bilirubin (BR), alpha-tocopherol (TOH), ascorbic acid (AA), and uric acid (UA) could be measured with good precision and recovery in the presence of human serum albumin (HSA). We measured the AOC of each antioxidant using a Cobas Mira S instrument (Roche Diagnostic Systems, Montclair, NJ) by measuring the inhibitory effect of a given compound on the oxidation of the radical cations of 2,2'-azino-di-(3-ethylbenzthiazoline 6-sulphonate) (ABTS) incubated with metmyoglobin and H(2)O(2). The assay had a linear AOC response range of 27-2,000 micromol/L. The within- and between-day coefficients of variation (CVs) did not exceed 3.4% and 4.2%, respectively. The AOC of albumin in serum is much greater than that of BR, TOH, AA, or UA owing to the substantially greater concentration of HSA in serum. An aliquot of a solution of AA, UA, BR, or TOH was added to HSA or distilled water, and the AOC was determined. The AOC of BR, TOH, AA, and UA increased in a linear way with increasing concentrations. However, we found that the magnitude of increase in the AOC of a mixture of HSA and any of these antioxidants was lower than the sum of the AOC of HSA and any one of the following: AA, UA, BR, or TOH (all expressed in micromol/L).